Plants perceive biotic stimuli by recognizing many different signaling compounds produced by the organisms with which they interact. Some of these substances have pathogen-associated molecular patterns generally acting as the triggers of defense reactions. They are perceived at low concentrations and comprise different structures including carbohydrates, proteins, glycoproteins, peptides, lipids and sterols (Hahlbrock et al. 2003: Proc Natl. Acad. Sci. USA 100 (supl 2), 14569-14576).
Microorganisms also synthesize and emit many volatile compounds with molecular weights less than 300 Da, low polarity, and a high vapor pressure (Schóller et al. 2002: J. Agrie. Food Chem. 50, 2615-2621; Schultz and Dickschat 2007: Nat. Prod. Rep. 24, 814-842; Splivallo et al. 2007a: Phytochemistry 68, 2584-2598). The contact with microorganisms or the plant defense reaction triggering agents does not only affect said defense reactions, but, very often, lead to photosynthesis reduction, and a transition from the source state (in which digestible organic compounds are produced) to the sink state (in which said digestible compounds are imported from tissues in which they are stored) (as a review, see Berger et al. 2007: J. Exp. Bot. 58, 4019-4026). An indication of the sink state in infected leaves is the up-regulation of the cell wall invertase, which results in the reduction of sucrose exportation from the infected leaf to other parts of the plant. In some cases, saccharolytic enzyme sucrose synthase (SuSy) is up-regulated after contacting microorganisms, which can serve to distribute the sucrose to the callus deposition and promote the biosynthesis of cell wall polysaccharides in the infection sites (Essmann et al. 2008: Plant Signaling & Behavior 3, 885-887). The contact with pathogens can also result in the down-regulation of genes involved in starch metabolism (Cartieaux et al. 2008: Mol. Plant-Microbe Interact. 21, 244-259; Fabro et al. 2008: Plant Physiol. 146, 1421-1439), which can make simple sugars available to the pathogen in the infection sites. These branched homoplysaccharides are synthesized by starch/glycogen synthase using ADPglucose (ADPG) as sugar donating molecule.
Starch and glycogen are the main storage carbohydrates in plants and bacteria, respectively, their mechanisms relate closely to that of amino acids by mechanisms which are still poorly understood. In Escherichia coli, amino acid deprivation triggers the response to stringent conditions, a pleiotropic physiological change switching the cell from a growth related mode to a maintenance/survival/biosynthesis mode. In conditions with limited nutrient (amino acids) supply, cell division stops and the demand for ATP-dependent proteins drops and in the nucleic acid synthesis and degradation. Excess ATP is then diverted from nucleic acid/protein metabolism towards glycogen biosynthesis if it is present in the medium with excessive carbon sources (Eydallin et al., 2007b: FEBS Lett 581, 2947-2953; Montero et al. 2009: Biochem. J. 424, 129-141). The typical sign of this pleiotropic physiological response is the accumulation of alarmone guanosine 5′-diphosphate 3′-diphosphate (ppGpp), a nucleotide which binds to the bacterial RNA polymerase to stimulate the expression of genes (included those involved in the metabolism of glycogen) expressed at the start of the stationary phase. The levels of ppGpp are controlled by RelA (a ppGpp synthase) and SpoT (a bi-functional enzyme showing ppGpp synthase and hydrolase activity) (Potrykus and Cashel 2008: Annu. Rev. Microbiol. 62, 35-51). E. coli mutants with damaged relA function, and the cells over-expressing spoT show a glycogen-deficient phenotype (Montero et al. 2009: Biochem. J. 424, 129-141). In contrast, E. coli mutants with damaged amino acid synthesis such as cysteine synthesis show a glycogen-excess phenotype as a result of the stringent response (Eydallin et al., 2007b: FEBS Left 581, 2947-2953). These mutants show a normal glycogen phenotype when they are cultured in medium supplemented with cysteine, which points to the existence of close connections between the metabolisms of sulfur, nitrogen and carbon.
Recent studies have shown that plants have a ppGpp-mediated regulating system similar to that in bacteria, which has been shown to play a crucial role in aspects such as plant fertility. ppGpp accumulates in the chloroplast of stressed leaves through the regulation of homologs RelA/SpoT (RSH) expression (Takahashi et al. 2004 Proc. Natl. Acad. Sci. USA 101, 4320-4324).
Starch degradation in plants is mainly hydrolytic, α-amylases and β-amylases playing important roles in endosperm and leafy cereals starch degradation, respectively (Scheidig et al. 2002: Plant J. 30, 581-591; Fulton et al. 2008: Plant Cell 20, 1040-1058) unlike in bacteria, where glycogen degradation occurs through the phosphorolytic pathway. From the initial demonstration that ADPG serves as precursor molecule for the biosynthesis of both bacterial glycogen and plant starch, the consideration that ADPG pyrophosphorylase (AGP) is the only enzyme catalyzing ADPG production has been rather widespread. The genetic evidence that bacterial glycogen biosynthesis occurs only through the AGP pathway (GlgC) has been obtained with glgC mutants. However, recent studies have shown that these mutants accumulate substantial amounts of glycogen and a normal content of ADPG. Furthermore, evidence has been provided demonstrating the existence of various important ADPG sources, different from GlgC, linked to the glycogen biosynthesis in different bacterial species.
Starch biosynthesis in leaves has generally been considered as occurring exclusively in chloroplast, and is segregated from the sucrose biosynthetic process occurring in cytosol (FIG. 1A). According to this classic view, starch is considered the end product of a unidirectional pathway in which AGP exclusively catalyzes ADPG synthesis, and works as the main regulating step of the starch biosynthetic process (Neuhaus et al. 2005: Trends Plant Sci. 10, 154-156; Streb et al. 2009: Plant Physiol. 151, 1769-1772). However, recent evidence has indicated the existence of an additional pathway in which ADPG linked to starch biosynthesis is produced de novo in the cytosol by means of SuSy. The sucrolytic enzyme SuSy is the main determinant of the sink strength intensively controlling the channelling of incoming sucrose towards starch and cell wall polysaccharides (Amor et al. 1995: Proc. Natl. Acad. Sci. USA 92, 9353-9357). It catalyzes the reversible conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate glucose and fructose. Although UDP is the preferred nucleoside diphosphate substrate so that SuSy produces UDPG, ADP also acts as an accepting molecule effective for producing ADPG.
According to this alternative view, both the sucrose and starch biosynthetic pathways closely interconnected by means of SuSy ADPG-producing activity (Muñoz et al., 2006: Plant Cell Physiol. 46, 1366-1376; Baroja-Fernández et al., 2009: Plant Cell Physiol. 50, 1651-1662), and by means of the action of a still to be identified ADPG translocator located in the membrane enveloping the chloroplasts. The “alternative” view of the starch biosynthesis in the leaves illustrated in FIG. 1B also assumes that both plastidial phosphoglucomutase and the AGP play an important role in removing glucose units derived from starch degradation.
Most of the studies on the plant-microorganism interactions have been carried out in conditions with physical contact between the host plant and the microorganism. However, little is known on how microbial volatile emissions can affect plant physiology in the absence of physical contact. However what is known is that microorganisms such as Pseudomonas spp., Streptomyces spp., Botrytis cinérea and different truffles produce ethylene (Splivallo et al. 2007b: New Phytologist 175, 417-424), a gaseous plant hormone which plays important roles in several aspects of plant growth and development, including seed germination, hypocotyl elongation, start of root hairiness, leaf and flower senescence, fruit ripening, starch accumulation, etc. Splivallo et al. (Splivallo et al. 2009: Plant Physiol. 150, 2018-2029) only recently provided evidence that the ethylene produced by truffles induces changes in Arabidopsis plant development, which are presumably accompanied by significant metabolism changes.
Regarding bacteria, the scarce work in which the effect of microbial volatiles on plant growth is described revolve around a limited number of specialized strains of plant growth promoting rhizobacteria (PGPR). Certain symbiotic bacteria existing in the ground and colonizing plant roots are called rhizobacteria. Most of the strains the culturing of which results in a positive effect on the growth of plants cultured in its presence, without the need of physical contact, belongs to the Bacillus genus or to the genus which is closely related to Bacillus, the Paenibacillus, to which bacteria belong which in the past were classified as belonging to the genus Bacillus. Therefore, it has been shown that volatiles emitted by rhizobacteria from strains belonging to the species Bacillus subtilis, Bacillus amyloliquefaciens or Bacillus cepacia, for example, promote Arabidopsis plant growth, facilitating nutrient uptake, photosynthesis and defense response, and reducing glucose sensitivity and the levels of abscisic acid (Ryu et al. 2003: Proc. Natl. Acad. Sci. USA 100, 4927-4932; Ryu et al. 2004: Plant Phylio 134, 1017-1026; Vespermann et al. 2007; Appl. Environ. Microbiol. 73, 5639-5641, Xie et al. 2009: Plant Signal. Behav. 10, 948-953). Specifically, Ryu et al. (Ryu et al. 2003: Proc. Natl. Acad. Sci. USA 100, 4927-4932) describe an increase in Arabidopsis thaliana seedling growth triggered by the organic volatiles released by specific PGPR strains, specifically Bacillus subtilis GB03 and Bacillus amyloliquefaciens IN937a, further commenting that their data demonstrate that the release of volatile organic compounds is not the common growth stimulation mechanism of all rhizobacteria. Since they are cultured in the amino acid-rich medium trypticase soy agar, both bacteria release 3-hydroxy-2-butanone(acetoin) and 2,3-butanediol, compounds not emitted by other tested PGPR the volatiles of which did not affect Arabidopsis growth, but which are also released by other bacterial strains for which capacity of increasing the germination and growth of plants such as Brassica olerácea without there being physical contact between plant and bacteria has been detected, such as the case of the Bacillus subtilis strain WG6-14 object of patent application US 2008/0152684 A1. However, there are many bacteria releasing these substances (some belonging to the Bacillus genus) which do not promote plant growth. As well as the mentioned GB03 and IN937a strains of the Bacillus genus, Ryu et al. only mention that the growth increase effect due to the release of volatiles will be detected for another one of the tested bacteria, Enterobacter cloacae JM22, although no data corroborating this result is shown nor the profile of volatile emitted by this bacterium is mentioned. Furthermore, an earlier article from the same research group (Ryu et al. 2004: Plant Phylio 134, 1017-1026) shows significant differences between the high capacity of the volatiles emitted by the two Bacillus strains for protecting the Arabidopisis thaliana plants from the effect of the pathogen Erwinia carotovora and the limited protective effect of the volatiles emitted by Enterobacter cloacae JM22.
Other strains of the Bacillus or Paenibacillus genera emitting volatiles capable of promoting the growth of different plants also have been described but, in these cases, the effect seems to be linked mainly to the capacity of controlling the growth of pathogens which are affecting the plant. This is the case, for example, of the bacillus Kyu-W63 described in Japanese Patent JP 10033064, the volatiles of which are capable of controlling the pathopoiesis due to the presence of fungi of the Cercospora genus in cucumber leaves, facilitating plant growth therewith. The description suggests that the effect could be similar by using other filamentous bacteria, provided that the culture is produced in a sugar-rich medium such as the PDA agar, a medium which is not defined in more detail; neither is proof demonstrating the influence of the suggested medium or the applicability of the method for any other filamentous bacterium provided. The method for increasing plant growth, based on compositions comprising a volatile metabolite produced by a bacterium which is claimed in the Korean Patent Application KR20090066412 also simultaneously relates to inducing protection against diseases and insect attacks and to promoting the growth of different plants, monocotyledons and dicotyledons. Examples of possibly useful metabolites include 3-acetyl-1-propanol, 3-methyl-1-butanol, indole, isoamyl acetate and butyl acetate. The abstract mentioned that the possible microorganisms which produce a volatile metabolite with the desired effect comprise bacteria belonging to the Bacillus or Paenibacillus genera, a strain of the species Paenibacillus polymyxa being the preferred microorganism.
As has been mentioned above, in addition to activating the defense system and promoting growth it has also been detected that the volatile compounds emitted by some bacteria have other effects in the plants. Therefore, Zhang et al. (Zhang et al. 2008: The Plant Journal 56, 264-273), describe how exposing Arabidopsis thaliana plants to the volatiles emitted by Bacillus subtilis GB03, again cultured in the culture medium trypticase soy agar, suppress plant glucose sensitivity, simultaneously causing a slight increase in sugar accumulation and an increase in photosynthesis, the latter being a process which is normally inhibited when the levels of soluble sugars accumulated in the plants increase. The plants contacting the volatiles emitted by B. subtilis GB03 show increases of 50-62% of the soluble sugar content with respect to the control plants which accumulate approximately 2 micromoles of hexose per gram of fresh weight (cf. FIG. 2, Zhang et al. 2008: The Plant Journal 56, 264-273). The increase in the soluble sugar content is generally associated with a reduction of the intracellular starch levels (Caspar et al. (1985) Plant Physiol. 79:11-17; Jones et al. (1986) Plant Physiol. 81: 367-371; Lin et al. (1988) Plant Physiol. 86:1 131-1135; Neuhaus and Stitt (1990) Plant 182:445-454; Szydlowski et al. (2009) Plant Cell 21, 2443-2457). Therefore, it is foreseeable that, in the conditions used by Zhang et al., the plants contacting the volatiles emitted by B. subtilis GB03 accumulate little starch. The method used by the group of Zhang et al. only allows measuring the content of glucose, fructose, fructose-6-phosphate and glucose-6-phosphate, although not the accumulated starch, although the absence of variations in the expression levels of genes involved in starch metabolism such as starch synthase or starch degrading enzymes shown by transcriptomic analysis of chloroplast proteins in plants exposed to the volatile shown in Supplementary Table 1 do not seems to indicate that an increase in this storage polysaccharide was foreseeable as the plant is subjected to the effects of the volatiles emitted by B. subtilis GB03. This interpretation is supported by the fact that the tests relating to the inhibition of hypocotyl height and seed germination indicate that the volatiles of B. subtilis GB03 do not cause a metabolic response to the treatment, since they do not seem to affect sugar metabolism, but do affect the sensitivity to said compounds.
According to what is known until now, all these effects on the plants are not common to the volatiles emitted by any bacteria. Therefore, for example, as discussed above, the tests performed by Ryu et al. (Ryu et al. 2003: Proc. Natl. Acad. Sci. USA 100, 4927-4932) demonstrate that several strains of Bacillus species, such as Bacillus pumilus T4 or Bacillus pasteurii C-9, as well as bacteria belonging to other genera such as Pseudomonas fluorescens 89B-61 or Serratia marcescens 90-166, were not capable of increasing the growth of Arabidopsis thaliana plants subjected to the effect of the volatiles emitted by said bacteria, despite the fact of having been equally cultured in the same culture medium, rich in sugars and amino acids: the trypticase soy agar. Another bacterium included in the same test, Escherichia coli DH5a, was used in the same test as control, since it is known as a strain which does not increase the growth of plants subjected to the action of the volatiles emitted by it.
Furthermore, it has also been shown that the volatiles of bacteria such as Pseudomonas spp., Serratia spp. and Stenotrophomonas spp., and of some species of fungi exert inhibitory effects on Arabidopsis plant growth (Splivallo et al. 2007b: New Phytologist 175, 417-424, Tarkka and Piechulla 2007: New Phytologist 175, 381-383)
Due to the lack of knowledge about how the microbial volatiles can affect the reprogramming of the cell metabolism, particularly primary carbohydrate metabolism, today it is not possible to act on the plant metabolism with microbial volatiles to promote its growth, since the mechanisms involved in promoting or inhibiting microorganism-activated growth mentioned above, or the conditions in which one or another is activated or the possible differences among microorganisms producing one effect or another are not clear. However, it would be interesting to know these mechanisms to enable designing a method for activating plant growth and/or flowering, and increasing the growth, biological and mechanical resistance thereof by means of using microbial volatiles and, preferably, for increasing starch synthesis in plants, since it is a product of great interest today in some industries. It would be particularly interesting for the method to be applicable to every type of plant and/or, particularly, to plants of agricultural interest in general, and which would be easy to apply. The present invention provides a solution to this problem.